LOCUS       Exported                5558 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pCS2 CMV expression vector driving V5-tagged mScarlet-I-fused human 
            KRAS4A.
ACCESSION   .
VERSION     .
KEYWORDS    JDW 836 (pCS2-V5-mScarlet-I-hsKRAS4A-WT)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5558)
  AUTHORS   Fish JE, Flores-Suarez CP, Boudreau E, Herman AM, Gutierrez MC, 
            Gustafson D, DiStefano PV, Cui M, Chen Z, Berman De Ruiz K, 
            Schexnayder TS, Ward CS, Radovanovic I, Wythe JD
  TITLE     Somatic Gain of KRAS Function in the Endothelium is Sufficient to 
            Cause Vascular Malformations that Require MEK but not PI3K 
            Signaling.
  JOURNAL   Circ Res. 2020 Jun 17. doi: 10.1161/CIRCRESAHA.119.316500.
  PUBMED    32552404
REFERENCE   2  (bases 1 to 5558)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5558
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        74..1058
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1093..1111
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1093..1110
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    1166..1190
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1209..1218
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1218..1259
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1275..1970
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1408..1426)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1676..1695
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(2596..2620)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(2643..2660)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    2665..2799
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2689..2708)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2743..2762
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2912..2932)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2912..2930)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2951..2967)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2951..2967)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2964..2986)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2975..2991
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(2999..3029)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3044..3065
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3182..3199)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3353..3941)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3433..3452)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4112..4972)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4735..4754
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4973..5077)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5103..5558)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5240..5261)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5452..5471
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gtcgaccata gccaattcaa tatggcgtat atggactcat gccaattcaa
      121 tatggtggat ctggacctgt gccaattcaa tatggcgtat atggactcgt gccaattcaa
      181 tatggtggat ctggacccca gccaattcaa tatggcggac ttggcaccat gccaattcaa
      241 tatggcggac ttggcactgt gccaactggg gaggggtcta cttggcacgg tgccaagttt
      301 gaggaggggt cttggccctg tgccaagtcc gccatattga attggcatgg tgccaataat
      361 ggcggccata ttggctatat gccaggatca atatataggc aatatccaat atggccctat
      421 gccaatatgg ctattggcca ggttcaatac tatgtattgg ccctatgcca tatagtattc
      481 catatatggg ttttcctatt gacgtagata gcccctccca atgggcggtc ccatatacca
      541 tatatggggc ttcctaatac cgcccatagc cactccccca ttgacgtcaa tggtctctat
      601 atatggtctt tcctattgac gtcatatggg cggtcctatt gacgtatatg gcgcctcccc
      661 cattgacgtc aattacggta aatggcccgc ctggctcaat gcccattgac gtcaatagga
      721 ccacccacca ttgacgtcaa tgggatggct cattgcccat tcatatccgt tctcacgccc
      781 cctattgacg tcaatgacgg taaatggccc acttggcagt acatcaatat ctattaatag
      841 taacttggca agtacattac tattggaagg acgccagggt acattggcag tactcccatt
      901 gacgtcaatg gcggtaaatg gcccgcgatg gctgccaagt acatccccat tgacgtcaat
      961 ggggaggggc aatgacgcaa atgggcgttc cattgacgta aatgggcggt aggcgtgcct
     1021 aatgggaggt ctatataagc aatgctcgtt tagggaaccg ccattctgcc tggggacgtc
     1081 ggagcaagct tgatttaggt gacactatag aatacaagct acttgttctt tttgcaggat
     1141 cccatcgatt cgaattcaag gatcaacaag tttgtacaaa aaagcaggct ggatccacta
     1201 gtgctagcgc caccatgggt aagcctatcc ctaaccctct cctcggtctc gattctacgg
     1261 gtggtgctgg tggtatggtg agcaagggcg aggcagtgat caaggagttc atgcggttca
     1321 aggtgcacat ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg
     1381 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
     1441 ccttctcctg ggacatcctg tcccctcagt tcatgtacgg ctccagggcc ttcatcaagc
     1501 accccgccga catccccgac tactataagc agtccttccc cgagggcttc aagtgggagc
     1561 gcgtgatgaa cttcgaggac ggcggcgccg tgaccgtgac ccaggacacc tccctggagg
     1621 acggcaccct gatctacaag gtgaagctcc gcggcaccaa cttccctcct gacggccccg
     1681 taatgcagaa gaagacaatg ggctgggaag catccaccga gcggttgtac cccgaggacg
     1741 gcgtgctgaa gggcgacatt aagatggccc tgcgcctgaa ggacggcggc cgctacctgg
     1801 cggacttcaa gaccacctac aaggccaaga agcccgtgca gatgcccggc gcctacaacg
     1861 tcgaccgcaa gttggacatc acctcccaca acgaggacta caccgtggtg gaacagtacg
     1921 aacgctccga gggccgccac tccaccggcg gcatggacga gctgtacaag tactcctccg
     1981 gactcagatc tggaggctcc ggaggcatga ctgaatataa acttgtggta gttggagctg
     2041 gtggcgtagg caagagtgcc ttgacgatac agctaattca gaatcatttt gtggacgaat
     2101 atgatccaac aatagaggat tcctacagga agcaagtagt aattgatgga gaaacctgtc
     2161 tcttggatat tctcgacaca gcaggtcaag aggagtacag tgcaatgagg gaccagtaca
     2221 tgaggactgg ggagggcttt ctttgtgtat ttgccataaa taatactaaa tcatttgaag
     2281 atattcacca ttatagagaa caaattaaaa gagttaagga ctctgaagat gtacctatgg
     2341 tcctagtagg aaataaatgt gatttgcctt ctagaacagt agacacaaaa caggctcagg
     2401 acttagcaag aagttatgga attcctttta ttgaaacatc agcaaagaca agacagagag
     2461 tggaggatgc tttttataca ttggtgaggg agatccgaca atacagattg aaaaaaatca
     2521 gcaaagaaga aaagactcct ggctgtgtga aaattaaaaa atgcattata atgtaaggcg
     2581 cgccctcgag agatctccca gctttcttgt acaaagtggt tgatcctctc gagcctctag
     2641 aactatagtg agtcgtatta cgtagatcca gacatgataa gatacattga tgagtttgga
     2701 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     2761 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     2821 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aattcgcggc cgcggcgcca
     2881 atgcattggg cccggtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     2941 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     3001 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     3061 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     3121 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     3181 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3241 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3301 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3361 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3421 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3481 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3541 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3601 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3661 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3721 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3781 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3841 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3901 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3961 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     4021 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     4081 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     4141 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     4201 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     4261 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     4321 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4381 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4441 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4501 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4561 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4621 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4681 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4741 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4801 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4861 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4921 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4981 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     5041 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     5101 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     5161 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     5221 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     5281 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     5341 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     5401 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     5461 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     5521 cacacccgcc gcgcttaatg cgccgctaca gggcgcgt
//
